1. Field of the Invention
The field of the invention is the modification of proteins by protein engineering. In particular, the invention concerns modified xylanase enzymes with improved performance at conditions of high temperature and pH. Xylanase enzymes are used to enhance the bleaching of pulp to make white paper. The invention enables xylanase enzymes to be produced with the benefits of enhanced bleaching associated with Family 11 xylanases, but with activity at higher temperature and pH conditions more suitable to the needs of a pulp mill's operation than xylanases currently available.
2. Brief Description of the Prior Art
Xylanase enzymes have been used commercially since 1991 to enhance the bleaching of pulp to make bright white paper. These enzymes are added to the pulp before the pulp is bleached, and remove a portion of the xylan in the pulp. This action allows the subsequent bleaching chemicals, including chlorine, chlorine dioxide, hydrogen peroxide, oxygen, ozone, and sodium hydroxide, to bleach the pulp more efficiently than in the absence of xylanase treatment. The enhanced efficiency of bleaching has allowed mills to reduce the amount of chlorine-based chemicals to use, which decreases the amount of toxic organochlorine compounds in the mill's effluent, as well as produce whiter pulp or allow the mill to save money on its bleaching chemicals. The commercial use of xylanase enzymes for bleaching has been reviewed by Tolan, et al, Pulp and Paper Canada, December 1995.
Xylanase enzymes have been reported from nearly 100 different microbes. The xylanase enzymes are classified into several of the more than 40 families of glycosyl hydrolase enzymes. The glycosyl hydrolase enzymes, which include xylanases, mannanases, amylases, beta-glucanases, cellulases, and other carbohydrases, are classified based on such properties as the sequence of amino acids, the three dimensional structure and the geometry of the catalytic site (Gilkes, et al, (1991) Microbiol. Reviews 55: 303-315).
Of particular interest for pulp bleaching applications are the enzymes classified in Family 11. All of these are xylanases and are known as the "Family 11 xylanases". Some publications refer to these synonymously as the Family G xylanases, but we shall use the term Family 11.
TABLE 1 lists the Family 11 xylanases known at the present time. Most of them are of molecular mass of about 21,000 Da. Three of the Family 11 xylanases- Clostridium stercorarium XynA, Streptomyces lividans XynB, and Thermomonospora fusca XynA-have a higher molecular mass of 31,000 to 50,000 Da. However, these xylanases have a catalytic core sequence of about 21,000 Da similar to the other Family 11 xylanases. The amino acid sequences of the Family 11 xylanases (or, for the larger enzymes, the catalytic core) show a high degree of similarity (FIG. 1). The Family 11 xylanases, which are of bacterial, yeast, or fungal origin, share the same general molecular structure (see FIG. 2, of CAMPBELL et al. U.S. Pat. No. 5,405,769).
TABLE 1 ______________________________________ Family 11 xylanases Microbe Xylanase ______________________________________ Aspergillus niger Xyn A Aspergillus kawachii Xyn C Aspergillus tubigensis Xyn A Bacillus circulans Xyn A Bacillus pumilus Xyn A Bacillus subtilis Xyn A Cellulomonas fimi Xyn D Chainia spp. Xyn Clostridium acetobutylicum Xyn B Clostridium stercorarium Xyn A Fibrobacter succinogenes Xyn C Neocallimastix patriciarum Xyn A Nocardiopsis dassonvillei Xyn II Ruminococcus flavefaciens Xyn A Schizophyllum commune Xyn Streptomyces lividans Xyn B Streptomyces lividans Xyn C Streptomyces Sp. No 36a Xyn Streptomyces thermoviolaceus Xyn II Thermomonospora fusca Xyn A Trichoderma harzianum Xyn Trichoderma reesei Xyn I Trichoderma reesei Xyn II Trichoderma viride Xyn ______________________________________
An enzyme is classified in Family 11 if it possesses the amino acids common to Family 11, including two glutamic acid (E) residues serving as the essential catalytic residues. These E residues are amino acids 86 and 177 by Trichoderma reesei xynII numbering. The corresponding location of the key E residues for other Family 11 xylanases is easily determined by aligning the amino acid sequences, a procedure familiar to those skilled in the art. The amino acids common to Family 11 xylanases are indicated in bold type in FIG. 1 (Wakarchuk, et al, Protein Science 3:467-475 (1994).
The Family 11 xylanases have several advantages over other xylanases in pulp bleaching applications. Most of the Family 11 xylanases are smaller than xylanases in other families. The small size relative to other xylanases is probably beneficial in penetrating the pulp fibers to release xylan from the pulp and enhance the bleaching. The Family 11 xylanases are also "pure" xylanases in terms of their catalytic activity. Unlike the xylanase enzymes in other families, these enzymes hydrolyze only xylan and do not hydrolyze cellulose. Cellulose hydrolysis damages the pulp and is unacceptable in a commercial mill. Among the Family 11 xylanases, the xylanases made by the wood-rotting fungus Trichoderma have been the most widely used in enhancing pulp bleaching. In particular, Trichoderma reesei xylanase II (Xyn II), with molecular weight 21,000 and isoelectric point 9.1, has been widely used.
In spite of the advantages of Family 11 xylanases in pulp bleaching, these enzymes have significant drawbacks. The range of temperature and pH that the enzymes exhibits activity on pulp are 45.degree. C. to 55.degree. C. and pH 5.0 to 7.5. A small proportion of mills have operated historically within these ranges. Typically, however, the pulp is at a temperature of 60.degree. C. to 70.degree. C. and a pH of 10 to 12. In some mills the adjustment of temperature and pH are acceptable and routine. However, in many mills achieving the desired treatment conditions causes severe problems.
Depending upon how the bleaching is carried out, cooling of the pulp to temperatures below 60.degree. C. can decrease the efficiency of bleaching to an unacceptable extent. For example, if a mill is bleaching entirely with chlorine dioxide and has a retention time of less than 20 minutes in the chlorination tower, the minimum temperature for adequate bleaching is 60.degree. C. If such a mill cannot heat the pulp between the enzyme treatment and the chlorination, which is often the case, then lower temperatures for the enzyme treatment stage are unacceptable.
Sulfuric acid is used to control the pH of the pulp. Depending on the metallurgy of the equipment, the use of sulfuric acid to control the pH can corrode the steel pipes and other equipment. Sulfuric acid is also a safety hazard.
Another minor problem with using these enzymes, and in particular Trichoderma reesei xylanase for bleaching applications is the low thermostability. There is the possibility that the warm ambient temperatures in the mills can inactivate the enzymes after several weeks storage. This problem is not as important as the difficulties of adjusting the temperature and pH of the pulp, but must be taken into account by using refrigerated storage or adding stabilizer compounds to the enzyme.
Therefore, the use of xylanase enzymes, particularly Family 11 xylanase enzymes, active at higher pH and temperature ranges than Trichoderma reesei Xyn II would be desirable. It would allow mills that operate outside of the active ranges of Trichoderma xylanase to be able to carry out xylanase treatment and obtain the benefits associated with the treatment. It would also allow mills to carry out xylanase treatment using less sulfuric acid and cooling water than is currently the case, saving production costs and increasing controllability and storage stability.
Before discussing the approaches that have been taken to improving the properties of xylanase enzymes, it is useful to define the following terms.
Thermophilicity is defined herein as the ability of an enzyme to be active at a high temperature. For example, xylanase #1 has more thermophilicity than xylanase #2 if it is capable of hydrolyzing xylan at a higher temperature than xylanase #2. Thermophilicity relates to enzyme activity in the presence of substrate. In the present invention, the substrate can be pulp xylan or purified xylan.
It is important to specify the substrate for purposes of defining the thermophilicity. Most xylanase enzymes are effective at higher temperatures in the hydrolysis of pure xylan than in the treatment of pulp. This is due to a combination of factors relating to the substrates (i.e. inhibitors present in the pulp) and to the length of time, pH, and other aspects of the procedures used to carry out the tests. Quantitative measures of thermophilicity refer herein to pure xylan substrates unless otherwise indicated.
Thermostability is defined herein as the ability of an enzyme to be stored incubated at a high temperature in the absence of xylan substrate, and then exhibit xylanase activity when returned to standard assay conditions. For example, xylanase #1 is more thermostable than xylanase #2 if it can be held at 70.degree. C. for 24 hours and retain all of its activity, while xylanase #2 loses all of its activity after 24 hours at 70.degree. C. In contrast to thermophilicity, thermostability relates to the enzyme activity remaining after incubation in the absence of xylan substrate.
These two terms are defined explicitly to overcome confusion in the literature, where the two terms are often used synonymously or to denote each other. Their present usage is consistent with Mathrani and Ahring, Appl. Microbiol. Biotechnol. 38:23-27 (1992).
Alkalophilicity is defined herein as the ability of an enzyme to be active at a high (alkaline) pH. For example, xylanase #1 has more alkalophilicity than xylanas #2 if it is capable of hydrolyzing xylan at a higher pH than xylanase #2. Alkalophilicity is analogous to thermophilicity and relates to enzyme activity in the presence of xylan substrate.
For improving xylanase for pulp bleaching applications, the thermophilicity and alkalophilicity are much more important than the thermostability. Most of the work described in the prior art has focussed only on improving the thermostability
Two generic approaches can be taken to make xylanase enzymes with higher pH and temperature ranges. These are: (1) screening naturally-occurring xylanase enzymes with the desired properties, and (2) using protein engineering to improve the properties of existing xylanase enzymes.
Among naturally occurring xylanases, thermostable enzymes have been isolated from thermophilic microbes, such as Caldocellum saccharolyticum, Thermatoga maritima and Thermatoga sp. strain FjSS3-B.1, all of which grow at 80.degree.-100.degree. C. (Luthi et al. 1990; Winterhalter et al. 1995; Simpson et al. 1991). However, all are relatively large in size with high molecular mass of 35-120 kDa (320-1100 residues). Some of these xylanases (C. saccharolyticum xylanase A) belong to families other than Family 11 and have both xylanase and cellulase activities (Luthi et al. 1990). Such cellulase activity is undesirable for pulp bleaching. Furthermore, hyperthermostable xylanases which function normally at extremely high temperatures have low activity at the comparatively lower temperatures for pulp bleaching.
Most of the Family 11 xylanases are effective in pulp bleaching applications at 45.degree. C. to 55.degree. C. However, Family 11 also includes at least two thermostable xylanases, both of which happen to have a higher molecular mass than the other Family 11 xylanases. These xylanases are Thermomonospora fusca xylanase (known as TfxA) of 296 amino acids and 32,000 Da (Irwin et al.(1994) Appl. Environ Microbiol. 60:763-770; Wilson et al. 1994, WO 95/12668) and Clostridium stercorarium xylanase A which is of 511 amino acids and 56,000 Da with an optimum temperature of 70.degree. C. (Sakka et al. (1993) Biosci. Biotech. Biochem. 57:273-277).
These thermostable xylanase enzymes have some features that are potential problems in pulp bleaching applications. First, the large molecular weight might limit the penetration of the enzymes into the pulp fibers. Second, these enzymes have at least a single copy of a cellulose binding domain (CBD) not present in the other Family 11 xylanases. The CBD, located in the extended C-terminus of TfxA, causes precipitation of the protein and loss of activity in storage.
Therefore, those naturally occurring xylanase enzymes have limitations.
An alternative approach is to carry out protein engineering of a well-known xylanase enzyme. By using protein engineering, specific changes can be made to th protein which might improve a desired property, such as temperature or pH range without compromising on secondary properties such as protein solubility.
When carrying out protein engineering to modify protein properties, one must select the general method to use and then the specific sites and modifications to make. The general methods include (1) site-specific mutagenesis, (2) random mutagenesis, (3) chimeric modification, (4) dimerization, and (5) glycosylation. Within each of these general methods, there are an enormous number of options of specific modifications to the protein that one can make. The effects of different mutations on enzyme characteristics, including thermophilicity and alkalophilicity, are often unpredictable. Generally, only a tiny fraction of all possible modifications, if any, provide significant benefit. Therefore, setting out to improve the properties of a protein by protein engineering is a difficult venture, and the limited success to date with Family 11 xylanases reflects this. The work with modified xylanases is described as follows.
Site-specific mutagenesis involves the modification of specific amino acids in a protein. The modifications based on site-specific mutagenesis are known as point mutations. Site-specific mutagenesis of Family 11 xylanases has been used to produce xylanase enzymes of slightly improved thermostability. CAMPBELL et al, (U.S. Pat. No. 5,405,769) described one manner of improvement of Bacillus circulans xylanase (abbreviated BcX), a xylanase of Family 11, through two types of modifications. These were (i) intramolecular disulfide bonds, and (ii) site-specific mutations at the N-terminus.
CAMPBELL et al, describes how disulfide bonds may be inserted between amino acids #98 and #152, #100 and #148, and # (-1) and #187, according to the amino acid numbering of Bacillus circulans xylanase. The disulfide modifications improved the thermostability of the xylanase at 620.degree. C. However, these disulfide-modified enzymes showed no gain in thermophilicity (Wakarchuck et al. (1994) Protein Engineering 7:1379-1386). Therefore, thermostability and thermophilicity are not necessarily coupled.
CAMPBELL et al, also describes three modifications (designated T3G, D4Y(F) and N8Y(F) ) near the N-terminus of BcX generated mutant xylanase with thermostability at 57.degree. C., a small increase of 2.degree. C. In the PCT publication WO 94/24270, which is related to CAMPBELL et al, there is a description of a fourth advantageous modification, S22P, for the improvement of BcX. This set of four modifications (designated TS19a in the document) showed a higher thermostability and thermophilicity than BcX. However, certain factors would limit the application of these modifications in Family 11 xylanases other than BcX. These mutations to convert residues-3, 4, 8 and 22 (BcX amino acid numbering) respectively into Glycine, Tyrosine (or Phenylalanine), Tyrosine (or Phenylalanine) and Proline, respectively, are irrelevant to the majority of the Family 11 xylanases, as they already possess these "good" residues (see FIG. 1). The best illustration of the inadequacy of these modifications is Xyn II of Trichoderma reesei, which possesses all four "good" residues, yet is mediocre in thermophilicity and alkalophilicity.
Random mutagenesis involves the modification of amino acids at random within the entire protein. This method was used to produce a Family 11 xylanase with improved thermostability by ARASE et al ((1993) FEBS Lett. 316:123-127), which described modest improvement of thermostability of a Bacillus pumilus xylanase (abbreviated BpX) through modifications at residues-12, 26, 38, 48 and 126 (according to the BpX amino acid numbering). However, ARASE et al did not report any improvement in the thermophilicity or alkalophilicity as a result of their particular modifications. The gain in thermostability by the most improved ARASE et al example in a BpX xylanase was small, only allowing the maintenance of 40% of the residual enzymatic activity after incubation at 57.degree. C. for 20 min. For two other BpX xylanases, with the modifications of residues 12 and 26 around the N-terminus, the gain in thermostability represented the maintenance of 1 and 11% residual activity after incubation, respectively. Furthermore, the BpX xylanase with the residue 26 modification has other modifications as well, so the contribution of this sole modification to thermostability, if any, is unclear from ARASE et al.
A chimeric modification involves substituting some of the amino acids of a protein with a sequence of amino acids from another protein. To our knowledge, such an approach has not been carried out with any Family 11 xylanases.
Dimerization involves combining two molecules into a single protein. This technique has been used to link two BcX molecules via an intermolecular disulfide bond (Wakarchuk, et al, Protein Engineering (1994)). The resulting dimeric BcX showed only an insignificant gain in thermostability, much less than BcX with an intramolecular disulfide bond described above.
It is well known that natural glycosylation, the attachment of carbohydrates to a protein, sometimes improves the thermostability of proteins, including in Trichoderma reesei xyn II. Synthetic glycosylation has not been used to improve these properties in a Family 11 xylanase.
No matter which method of protein engineering is used, a key aspect is determining which amino acids to modify, because few choices will improve the properties of the enzyme. This point is illustrated by the work of Sung, et al, Biochem. Cell Biol. 73:253-259 (1995), who modified amino acid #19 in Trichoderma reesei xylanase II from asparagine to lysine. This modification decreased the thermophilicity of the enzyme by 3.degree. C.
Therefore, in spite of a large amount of effort with Family 11 xylanases, there has not yet been a modified Family 11 xylanase produced with significantly improved thermophilicity and alkalophilicity. Such an enzyme, and in particular an engineered version of Trichoderma reesei xyn II, would have immediate application to the commercial process of producing bleached pulp with decreased requirements for bleaching chemicals while meeting the process conditions of the mills. Such an enzyme would also have potential application in other areas. Some examples of these are as animal feed additives to aid in the digestibility of feedstuffs, where high temperature pelleting makes current enzymes unsuited in many cases; and the processing of wheat and corn for starch production, in which the high temperatures destroy current enzymes.